|Publication number||US3929813 A|
|Publication date||Dec 30, 1975|
|Filing date||Dec 26, 1973|
|Priority date||Dec 26, 1973|
|Publication number||US 3929813 A, US 3929813A, US-A-3929813, US3929813 A, US3929813A|
|Inventors||Nicolae S Bodor, Takeru Higuchi, Efraim Shek|
|Original Assignee||Interx Research Corp|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (6), Referenced by (39), Classifications (10)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent 1 Higuchi et al.
[ 51 Dec. 30, 1975 154] NOVEL PRO-DRUG DERIVATIVES OF PYRIDINIUM ALDOXIME TYPE CHOLINESTERASE REACTIVATORS AND PROCESS FOR PREPARING SAME  Inventors: Takeru Higuchi; Nicolae S. Bodor;
Efraim Shek, all of Lawrence, Kans.
 Assignee: Interx Research Corporation,
221 Filed: Dec. 26, 1973 21 Appl. No.1 428,215
 US. Cl. 260/296 M; 424/263  Int. Cl. C07D 211/26  Field of Search 260/296 M  References Cited UNITED STATES PATENTS 2,816,113 12/1957 Wilson et al. 260/296 M 3,054,798 9/1962 Hackley et al. 260/296 M 3,285,927 ll/l966 Ellin 260/296 M 3,468,896 9/1969 Myers et a1... 260/296 M 3,629,425 12/1971 Hussain 260/296 M 3,773,775 11/1973 Hagedorn 260/295.5 A
FOREIGN PATENTS OR APPLICATIONS 889,074 2/1962 United Kingdom 260/296 M OTHER PUBLICATIONS Klingsberg, Pyridine and Its Derivatives, part 2, pp. 46 to 51 and part 4, p. 173, Interscience Publishers (1961 for part 2 and 1964 for part 4).
Chemical Abstracts, Sixth Collective Index, pp. 9826 s to 9827 s, (1966).
Chemical Abstracts, Eighth Collective Index, 2604 S to 2606-S, (1973).
Chemical Abstracts, Seventh Collective 19,2668 to 19,267S, 1970 Index,
Primary ExaminerJohn D. Randolph Attorney, Agent, or Firm-Charles N. Blitzer  ABSTRACT There is provided, novel pro-drug forms of pyridinium aldoxime type cholinesterase reactivators, namely, dihydropyridinium aldoximes, having the formula:
wherein R represents a member selected from the group consisting of an alkyl (C -C group, a
wherein Z represents a member selected from the group consisting of a CH CH group, a CH- OCI-I group, a CH CI-I OCH,CH group, and a -CI-I OCI-I CI-I -OCI-I wherein R represents a member selected fromkhe group consisting of a hydrogen atom, a methyl group, an acyl group and a group; and wherein X represents an anion derived from a pharmaceutically acceptable acid addition salt.
These compounds are useful in reactivating cholinesterase, inhibited following exposure to and/or ingestion of conventional anti-cholinesterase agents, especially in the brain.
22 Claims, No Drawings group;
NOVEL PRO-DRUG DERIVATIVES OF PYRIDINIUM ALDOXIME TYPE CHOLINESTERASE REACTIVATORS AND PROCESS FOR PREPARING SAME BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION The present invention is directed to novel pro-drug forms of cholinesterase reactivators, and more specifically, the present invention is directed to pro-drug forms of pyridinium aldoxime type cholinesterase reactivators. A most representative member of this class of cholinesterase reactivators is l-methyl-pyridinium-2- aldoxime iodide (or chloride or methanesulfonate), hereinafter referred to as 2-PAM.
As employed in this application, the term pro-drug" refers to a derivatized form of a proven drug, for example, 2 PAM, which when administered to an individual, is enzymatically oxidized in the bloodstream, to the extent that the proven drug (2-PAM) is released at its therapeutic site or sites of activity and especially the brain.
2. DESCRIPTION OF THE PRIOR ART 2-PAM, chemically known as 1-methylpyridinium-2- aldoxime is a white crystalline, water-soluble powder, usually employed in the chloride form, having the following formula:
Conventionally, the compound is employed in the form of its acid addition salt, such as, the chloride salt, the iodide salt, or the methanesulfonate salt.
Intoxication by anti-cholinesterase compounds may occur following accidental exposure to organophosphorus insecticides and other toxic chemical agents or even after administration of excessive amounts of drugs like neostigmine, normally employed for the management of glaucoma or myasthenia gravis. The pharmacologic effect of anti-cholinesterases is chiefly due to the inhibition of cholinesterase enzymes throughout the body. The search for satisfactory antidotes to counteract the effect of such toxic agents has led to a series of oxime derivatives of which 2-PAM appears to be the compound of choice, namely because of its physiologic compatibility, its excellent water solubility and its high oxime content per mole of compound. The most striking characteristic of the oxime is that it reactivates cholinesterase, which becomes inhibited by the anticholinesterase agent.
The pharmacologic effects of anti-cholinesterases, such as nerve agents (GB and VX), organophosphorus insecticides (Parathion), and quaternary ammonium compounds (neostigmine), may be ascribed to the inhibition of cholinesterase enzymes in the tissues, thereby resulting in cholinergic effects caused by the accumulation of acetylcholine in the effector organs. The chief actions of these compounds can be classified as follows:
1. Muscarine-like effect, manifested by nausea, vomiting, abdominal cramps, diarrhea, sweating, increased salivation, bronchial secretion, and bradycardia;
2 2. Effect on the central nervous system, characterized by anxiety, headache, and sometimes ataxia, coma, and convulsions; and
3. Nicotine-like effects, including as the most important one, muscular paralysis.
Severe poisoning by anti-cholinesterases results in paralysis of respiration by both central and peripheral effects, with consequent death unless prompt therapy is instituted.
While large doses of atropine abolish the muscarinelike effect, and partially alleviate the central nervous system manifestations of intoxication by the anticholinesterase compounds, the peripheral neuromuscular blockade (nicotine-like effect) is not appreciably diminished. See, R. V. Brown, et al., J. Pharmacol. Exp. Therap., 120, 276, (1957); D. Grob, Arch. Internal Med., 98, 221, (1956); and H. M. Kunkel, et al., Proc. Soc. Exp. Biol. Med., 92, 529, (1956).
The neuromuscular blockade, attributable to the accumulation of acetylcholine at the motorend-plate, persists until partial restoration of cholinesterase activity of the muscle can be brought about. The effectiveness of oximes in treating anti-cholinesterase intoxication is attributable largely to their ability to overcome the neuromuscular blockade. The reversal of the neuromuscular blockade produced by alkyl phosphates is believed to be due to reactivation of muscle cholinesterase. See, D. Grob, and R. J. Johns, Am. J. Med., 24, 497, (1958).
Many workers have demonstrated that cholinesterase inactivated by organophosphorus compounds may be reactivated in vitro" by oximes. In studies with experimental animals, the actions of anti-cholinesterase agents on smooth, cardiac and skeletal muscles could be reversed by oximes, and in addition, their lethal effects reduced, although the effectiveness of the oximes will vary considerably in different species. See, B. M. Askew, Brit. J. Pharmacol., 11, 417, (1956); R. V. Brown, J. Pharmacol. Exp. Therap., 120, 276, (1957); R. Holmes, and E. L. Robins, Brit. J. 'PharmacoL, 10, 490, (1955); H. Kewitz, et al., Arch. Biochem. Biophys., 64, 456, (1956); and J. H. Wills, et al., Science, 125, 743, (1957).
In man, the intravenous dose of 2-PAM (as the iodide) required to alleviate the toxic effect of a nerve agent, such as G. B. (Sarin) has been reported to be in the range of 14 to 28 mg./Kg. of body weight. See, D. Grob, and R. .1. Johns, Am. J. Med., 24, 497, (1958). Consequently, since 1.4 g. of the iodide are equivalent in oxime content to 1 g. of the chloride, the corresponding intravenous dose of 2-PAM chloride is 10 to 20 mg./l(g. of body weight. See, A. A. Kondritzer, et al., J. Pharm. Sci., 50, 109, (1961).
Zvirblis has studied the distribution of 2-PAM in serum and tissues of rabbits. The greatest concentration of 2-PAM found in the kidney. In man, 2-PAM is eliminated primarily via the kidney. See, P. Zvirblis, unpublished data, US. Army Chemical Research and Development Laboratories. Following an intravenous dose of 15 mg. of 2-PAM per Kg. of body weight, the half-life in man has been found to be about 0.8 hours. See, B. V. Jager, and G. N. Stagg, Bull. Johns Hopkins Hosp., 102, 203, (1958). Approximately of the compound was excreted during the first 6 hours, and most of the unchanged oxime was found in the urine within the first 30 minutes. 2-PAM was not found to be bound appreciably to human serum protein, nor for that matter, to enter the erythrocytes.
Because 2-PAM is an ionic compound (quaternary ammonium salt), it is not expected to cross the bloodbrain barrier, although there is conflicting evidence regarding this question. But the most recent evidence indicates that some crossing of the blood-brain barrier does occur in the rat within ten minutes after administration of Z-PAM. Moreover, in patients intoxicated by Parathion, 2-PAM effected rapid recovery of consciousness. See, A. J. Funckes, Arch. Env. Health, 1, 104, (1960); T. Nanba, and K. Hiraki, J. Am. Med. Assoc., 166, 1834, (1958); and A. Schuchter, et al., Arzneimittel Forschg, 10, 399, (1960). The oxime was found to have significant anti-convulsant action in some patients, this finding supporting the view that oximes do cross the blood-brain barrier.
However, even with such encouraging results, it is well felt among those individuals familiar with 2-PAM that if the same does, in fact, cross the blood-brain barrier, the amount which crosses the barrier is extremely small. Thus, an LP. (intraperatoneal) dose of 100 mg./l(g. of 2-PAM reactivated only 4% of the cholinesterase in the brain of the rat after intoxication in vivo by Sarin. See, J. P. Rutland, Brit. J. PharmacoL, 13, 399, (1958).
It is speculated that since some body tissues exhibit a pH as high as 8.5 (the pKa of the oxime part of 2-PAM) such a high pH would permit some 2-PAM to dissociate and pass through the blood-brain barrier as a zwitter cH=No but only in a small amount which would not achieve therapeusis. Experiments also show that 2-PAM does not penetrate all areas of the brain to the same extent, but the test'animals are still protected from the lethal effects of paraoxon. See, F. Hobbiger, and V. Vojvodic, Biochem. PharmacoL, 16, 455, (1967).
Furthermore, more recent publications disclose that if a therapeutic amount of 2-PAM fails to reach the brain site immediately, the bound cholinesterase cannot be subsequently regenerated by oximes, i.e., its regeneration is irreversible. This process, called aging, occurs at different rates, mainly depending on the nature of the poison, i.e., organophosphorus compound. See, E. Heilbronn, Svensk kemisk tidskr., 77, 1 1, (1965). For example, cholinesterase inhibited by DFP (diisopropyl phosphorofluoridate) is transformed into the non-reactivatable form in a few hours, while aging of a soman inhibited enzyme occurs in 2 minutes. See, W. D. Erdmann, Naunyn-Schmiedebergs Arch. Pharmak., 263, 61, (1969).
As to oral administration of 2-PAM, while the same can be and has been administered orally, the amount of absorption of 2-PAM into the bloodstream amounts to only some 20 to 30 percent. Consequently, the oral route of administering 2-PAM (5 g. initially), in time of need, is less desirable than any of the conventional parenteral approaches, i.e., intravenous, intraperitoneally, or intramuscularly, except for prophylactic use or maintenance in certain intoxications, for example, Parathion. See, H. Edery and G. Schatzberg Porath, Arch. Int. Pharmacodyn., 121, 104, (1959); A. A. Kondritzer, P. Zvirblis, A. Goodman, and S. H. Paplamus,
4 J. Pharm. Sci., 57, 1142, (1968); F. R. Sidell, N. A. Groff, and R. J. Ellin, ibid, 58, 1093, (1969); F. R. Sidell and N. A. Groff, ibid, 60,860, (1971); F. R. Sidell, N. A. Groff, and A. Kaminskis, ibid, 61, 1136, (1972); respectively.
In summary then, 2-PAM, while useful to some extent in overcoming the effects of anti-cholinesterase agents, still leaves much to be desired in terms of( 1 its inability to appreciably pass through the blood-brain barrier, (2) its extremely poor bio-availability, and (3) its unfavorable distribution, poor retention rate in the body, and/or no protein binding.
SUMMARY OF THE INVENTION Accordingly, it is one object of the present invention to provide novel pro-drug forms of 2-PAM and related pyridinium aldoximes, which when placed in the bloodstream of a warm-blooded animal exists in a non-ionic form so as to also permit passage of same through the blood-brain barrier, after which, the compound is enzymatically oxidized to release 2PAM or the corresponding pyridinium aldoxime at its therapeutic site or sites of activity.
It is a second object of the present invention to provide novel compounds as described above which when administered orally to a warm-blooded animal, will be absorbed through the intestinal tract and introduced into the bloodstream at high bio-availability levels, after which enzymatic oxidation will occur to release 2-PAM or a corresponding pyridinium aldoxime at its therapeutic site or sites of activity.
Finally, it is a third object of the present invention to provide novel compounds as described above, which when placed in the bloodstream of a warm-blooded animal, via any route of administration, will pass through the blood-brain barrier, be enzymatically oxidized to 2-PAM or to the corresponding pyridinium aldoxime, thus permitting the same to act therapeutically at all body sites of need, including the brain, and yet, be retained by the body in such a manner as to be eliminated at a rate commensurate with therapeusis. That is, substantial protein binding will occur, before its transformation into 2-PAM or the corresponding pyridinium aldoxime.
Accordingly, all of the foregoing objects are achieved by administering to a warm-blooded animal, e.g., a mammalian species, by any suitable route of administration (orally, intravenously, intramuscularly, or intraperitoneally), a compound of the formula:
wherein R represents a member selected from the group consisting of an alhyl (C -C group, a
wherein z represents a member selected from the group consisting of a -CH CH group, a CH OCH group, a CH CH OCH CH group, and a CH OCH CH OCH group; wherein R represents a member selected from the group consisting of a hydrogen atom, a methyl group, an acyl group and group; and wherein X represents an anion derived from a pharmaceutically acceptable acid addition salt.
These compounds are useful in reactivating cholinesterase, inhibited following exposure to and/or ingestion of conventional anti-cholinesterase agents, especially in the brain.
With reference to the generic formula for the pro-2- PAM and related pyridinium aldoxime compounds of this invention, X represents an anion derived from a pharmaceutically acceptable acid addition salt. The phrase pharmaceutically acceptable acid addition salt" as used herein, refers to non-toxic pharmaceutically acceptable acid addition salts formed from nontoxic inorganic or organic acids. For example, but without limitation, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and the like; and the salts prepared from organic acids, such as acetic, propionic, succinic, glycollic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, fumaric, toluenesulfonic, and the like.
DETAILED DESCRIPTION OF THE INVENTION The term warm-blooded animal as employed in this application refers to any warm-blooded animal, though the mammalian species is preferred, since it is the species most often affected by anti-cholinesterase agents.
As mentioned earlier, 2-PAM is an ionic species; that is, it is a quaternary salt, and because of this ionic feature, Z-PAM cannot readily pass through the bloodbrain barrier. As such, therapeutic levels of 2-PAM in the brain cannot be achieved.
Quite uniquely, the present inventors approached the blood-brain" barrier impediment of 2-PAM from a unique standpoint. They first realized that 2-PAM derivatives had to be developed, which (1 would be non-ionic when they reached the bloodstream, and (2) would be.subsequently enzymatically oxidized to release 2-PAM in atherapeutic amount at the bodys site or sites of need, and especially,'the brain. To this end, they developed pro-drug formsof 2-PAM and related compounds which uniquely contain a tertiary nitrogen atom, exhibitinga low basicity, Le, a pKa below 7.4, and thus a pKa of 6.32 in the case of the pro-2-PAM. On this basis, the skilled artisan can readily appreciate that such compounds, when introduced into the bloodstream of a warm-blooded animal whose pH is about 7.4 would remain essentially non-ionic, thus permitting the pro-drug form to be highly protein bounded and/or enter the erythrocytes and thus to pass through the blood-brain barrier and be subsequently enzymatically oxidized to release 2-PAM or a related pyridinium aldoxime in high bio-availability levels at the bodys therapeutic site or sites of need. In this vein, the compounds incompassed by the above-generic formula function in the manner described above. In addition to containing a tertiary nitrogen atom having a pKa of about 6.32, and thus, essentially, non-protonated at the pH of plasma, the observer will readily appreciate the fact that the compounds of this invention are not quaternary salts. As such, they readily pass the blood-brain barrier.
Wiith respect to the salt feature (acid addition salt) of the compounds of this invention, the salt form is employed for the sole purpose of rendering stability to the pro-drug free base form, prior to administration. That is, once the pro-drug compounds of this invention are administered by any route, the salt moiety is cleaved, thus releasing the remaining tertiary amine form, which readily transcends the blood-brain barrier. Next, the pro-drug is oxidized enzymatically to 2-PAM or the related pyridinium aldoxime in an analogous manner to the nicotinamide dinucleotide (NAD) co-enzyme mediated oxide-reduction system; where the co-enzyme, in turn, is reduced to the dihydro from (NADH). For details about this enzymatic oxidoreductive system see, U. Eisner and -J. Kuthan, The Chemistry of Dihydropyridines, Chem. Rev., 72, 1 (I972).
It should be emphasized that in one instance, a prodrug form of this invention will release 2-PAM in the bloodstream following enzymatic oxidation. In the other instances, related pyridine aldoxime derivatives will be released. In any event, the ultimate pyridine aldoxime compound released (other than 2-PAM) will function in the same manner as 2-PAM to reactivate bound cholinesterase.
While all the compounds of this invention will achieve the objectives previously outlined, certain compounds are preferred as follows: I
l l-methyl-l ,6-dihydropyridine-Z-aldoxime and 1 its HX salts, wherein X represents a pharmaceutically acceptable anion.
2. Trimethylene-bis-[ l-(l,6- or 1,4-dihydropyridine- 4-aldoxime] and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
3. Bis-(4-hydroxyiminomethyl-l,6- or -l,4-dihydropyridine-l-methyl) ether and its HX salts, wherein X represents a pharmaceutically acceptable anion.
4. Bis-(2-hydroxyiminomethyl-l,-dihydropyridinel-methyl) ether and its HX salts, wherein X represents a pharmaceutieally acceptable anion.
5. Bis-[2-(4-hydroxyiminomethyl-l,6- or l,4-dihydropyridino) ethyl] ether and its HX salts, wherein 5 The compounds of this invention can be prepared according to the following reaction scheme:
@ 1 1) NaBH 2) HCN CH=NOR 5,6-dihydropyridinium salt Type A The first two steps are carried out at standard tem- 3,6-dihydropyridinium salt In the last step, elimination of HCN is carried out at standard temperature and pressure, under a standard N atmosphere, in a C -C aliphatic alcoholic solvent, methanol being preferred, for several hours (preferably 1 to 2 hours). During the reaction, the starting material is dissolved and a new product, the final dihydropyridinium salt is precipitated. Depending on the position of the oxime and of the CN groups in the starting material, the salt of a 1,6- or l,4-dihydropyridine derivative is obtained.
As all the salts exist in an enamine form, the double bond(s) of the obtained dihydropyridine ring will rearrange during the salt formation, i.e., during the elimination process. Thus, the following basic enamine salt types (isomers) are possible:
NC@ CH=NOR 3/ CH=NOR ium salt yp B Type C It is well known that the relative energy difference perature (preferably at C.) and standard pres- 5 between dihydropyridine isomers is small (less than 4 sure in an N atmosphere, using a reducing agent, such as any metal hydride, sodium dithionite, but preferably sodium borohydride (NaBl-I and a good nucleophile, preferably HCN (formed from NaCN and HCl in a homogeneous solvent system (e.g., water, waterther the l,2,3,6, or the l,2,3,4, depending on the position of the oxime type group, and is crystallized from a suitable solvent, preferably from ether.
CH=NOR HX/THF The third step is carried out at standard temperature and pressure, using an ether type solvent (ethyl ether, dioxane, tetrahydrofuran), but preferably tetrahydrofuran (THF) saturated with anhydrous HX (X is de- Kcal/mole). Thus, they can usually rearrange into another, on the simple action of a solvent.
For any skilled artisan it will be obvious that the position of the double bonds in the above types is controlled both kinetically and thermodynamically. However, from the point of view of the present invention, i.e., the ease to be oxidized, back to a pyridinium ring, the question as to which isomer will be obtained, is absolutely irrelevant. All three possible isomers have the same value from the view of the present invention, and they are all considered as part of the basic idea of this invention.
All steps of the synthesis are carried out under vigorous non-oxidizing conditions in order to prevent premature oxidation of the dihydropyridine derivatives into pyridinium form. In step (I), an equimolar ratio of fined as set forth earlier, but preferably Cl). The salt ,0 reactants can be employed. In step (2), an equimolar formed is collected by filtration, after stirring the mixture for about A hour and cooling.
NC 1 1 HCN ratio of reactants can be employed, though an excess of HCN is preferred. In step (3), an equimolar ratio of H=NOR CH=NOR 3,4-dihydropyridin- 9 reactants can be employed,though an excess of HX is preferred.
The uniqueness of the process employed to prepare the compounds of this invention resides in the fact that first, the reduction step is stoppedafter reduction of only one double bond (addition of two hydrogen atoms) of the pyridine ring, by addition of a suitably good nucleophile leaving group (HCN, HSCN, etc.) on one of the double bonds of the dihydropyridine derivative formed. Otherwise, reduction could not be stopped at the dihydro stage and various tetrahydro derivatives would form which cannot be transformed back to the necessary dihydro derivatives. (It is known that tetrahydropyridinium oximes are inactive as cholinesterase reactivators).
Secondly, the cyano adduct formed is a relatively stable intermediate which can be well stabilized in its protonated salt form, which, on the other hand can be transformed easily into the desired dihydropyridinium salt, by elimination of an HCN molecule, using the conditions described earlier.
These compounds can be administered parenterally (l.V., l.P., or [.M.) by simply combining a therapeutic effective amount of same with any pharmaceutically acceptable inert parenteral carrier, and with respect to oral administration, since the compounds of the present invention are absorbed effectively through the intestine, and on the other hand, being highly sensitive compounds, they must be administered in a therapeutic amount by means of an inert enteric coated medium. With respect to eigher dosage form, parenteral or oral, it should be emphasized that the carrier material, in either case, must be inert (non-oxidizing) in nature. The reason for this, is that if the compounds of this invention are contacted with an oxidizing agent prior to entrance into the bloodstream, the compound will be oxidized to 2-PAM or the like aldoxime compound immediately, and as such, the purpose and objectives of the present invention would be defeated. While detailed examples will be provided later in the application, demonstrating the preparation and administration of a parenteral and oral dosage form suitable for this invention, the ordinary skilled artisan can readily acquaint himself with such forms by referring to the text entitled REMINGTONS PHARMACEUTICAL SCI- ENCES," Fourteenth Edition (1970), pages 1519-1544 and 1689-1691, respectively.
A better understanding of the present invention will be gained from the following examples, which are simply intended to be illustrative and non-limitative of the present invention.
EXAMPLE 1 (Preparation of l-methyl-l ,6-dihydropyridine-Z-aldoxime hydrochloride) Firstly, the precursor compound, l-methyl-Z-cyanol,2,3,6-tetrahydropyridine-2-aldoxime was prepared.
To a solution of 76 g. (1.55 mol) of sodium cyanide in 300 ml. of freshly distilled water, saturated with nitrogen, there was added 1 14 ml. of 18% hydrochloric acid. The aqueous solution was layered with 1000 ml. of distilled ether and cooled to 15 C. One hundred (100) g. (0.38 mol) of 2-pyridine aldoxime methiodide (2-PAM) was added to the cold solution. Subsequently, 19 g. (0.495 mol) of sodium borohydride (NaBl-l was added and the solution was warmed to 25 C and kept at that temperature for hours with vigorous stirring.
The ether layer was separated and the water layer was filtered, the precipitate being extracted with ether. The combined ether layers were evaporated. The precursor compound was purified by washing with 30% diethyl ether in cyclohexane. The yield of the precursor compound was 44.5 g. (71%), and the melting point of same was 1 121 15 C (decomp). The NMR spectrum was consistent with the structure of the compound obtained. Analysis calculated for C H ON C, 58.15; H, 6.67; and N, 25.45. Found: C, 58.34; H, 7.01; and N, 25.22. IR. spectra: Weak, 1630 cm", 1665 cm: C=C, C=N; Strong: 955 cm: NO; 2740 cm, 1445 cm: N-CH 3180 cm: O-H.
Subsequently, 200 ml. of tetrahydrofuran (peroxide free) was saturated with hydrogen chloride gas. Fifteen (15) g. of the precursor compound was added and the mixture was stirred for 30 minutes. The mixture was then cooled and the precipitate was collected, washed with ether, and dried in vacuo. In this manner, the hydrochloride form of the precursor compound was obtained in a yield of 17.5 g. (95%), mp 127-130 C (decomp). Analysis calulated for C H ON Cl: C, 47.64; H, 5.96; N, 20.84. Found: C, 47.39; H, 5.93; N,
20.57. IR. spectra: (KRr pellet) -N -H at 2260 cm (broad); Weak: 1640, 1675 cm: C=C, C=N; Strong: 970 cm: NO; 3180 cm": OH.
To 200 ml. of cooled, but freshly boiled methanol, 17.0 g. (0.084 mol) of the hydrochloride form of the precursor compound (l-methyl-2-cyano-1,2,3,6-tetrahydropyridine hydrochloride) obtained earlier was added. The suspension was mixed under nitrogen for 1% hours. During that time, a solution was formed and a new product was precipitated. The product was filtered, washed with ether, and dried in vacuo to yield 11.7 g. of the objective compound (l-methyl- 1,6-dihydropyridine-2-aldoxime hydrochloride), mp 179 C (decomp). The NMR spectrum was consistent withthe structure of the compound obtained. The analysis of the compound by l. R. (KBr. pellet) showed strong and medium bands at 1655, 1700. U.V. analysis in 0.1 N HCl-25l nm (Fl3,300); pH of 8 308 nm; 0.1N NaOH-260 nm (F7640) and 340 nm (e -5440).
Analysis calculated for C H N OCl: C, 48.14; H, 6.30; and N, 16.04. Found: C, 47.86; H, 6.59; and N, 15.97.
By following the procedure outlined above for Example l and using the appropriate reactants, the remaining compounds of the present invention can easily be obtained.
The pKa of the tertiary amine was determined to be 6.32 i 0.06 by titration and U.V. methods. The pKa of the oxime moiety is about 10.5 as determined by the U.V. noted above.
EXAMPLE 11 IN VIVO CHOLINESTERASE REACTlVATlON STUDIES 1 -methyl-1 ,6-dihydropyridine-2-aldoxime hydrochloride (the compound of Example I), noted as DPAM was evaluated for its ability to protect against anti-cholinesterase agent toxicity, commensurate with the prescribed procedure set forth by R. A. Lehman and M. E. Nicholas, Proc. Soc. Expt. Biol. Med., 104, 550 (1960). Specifically, an anti-cholinesterase agent, phospholine iodide was injected l.P. (intraper itoneally) at twice the lethal dose into white mice of male sex, weighing about 30.0 plus or minus 3.0 g. Four groups of 10 mice each were studied as outlined below in Table 1.
1 1 When using 2-PAM, the same is injected I.P. immediately following phospholine iodide administration. However, when I-methyl-1,6-dihydropyridine-2-aldoxime hydrochloride was employed, the same was injected 10 minutes after the mice were closed with phos- 5 pholine iodide.
12 in the plasma, practically none of the drug being observed in the blood cells or blood proteins. This is obvious from the data set forth above, since the volume of plasma is approximately one-half of the blood. Therefore, the plasma concentrations of 2-PAM chloride are twice that of the concentration of 2-PAM chloride in TABLE I GROUP 2-PAM CI DPAMJ-ICI PHOSPHOLINE SALINE SURVIVED IODIDE l mg./cc. l0 mg./cc. 0.1 mg./cc.
I 0.5 mg./kg. 0.2 cc 1/10 (twice the LD ll 50 mgJKg. 0.5 mg./Kg. lO/IO (twice the LB Ill 50 mg./Kg. 0.5 mg./Kg. IO/IO (twice the LD IV 50 mgJKg. 0.2 cc /10 the whole blood.
EXAMPLE III (Z-PAM CHLORIDE AND l-METI-IYL-l,6-Dll-IYDROPYRIDlNE-Z-ALDOX- [ME-HYDROCHLORIDE CONCENTRATIONS IN BLOOD AFTER I.V. ADMINISTRATION) In accordance with the procedure outlined by J. R. May, P. Zvirblis, and A. A Kondritzer, J. Pharm. Sci., 54, 1508 (I965), Beagle dogs were injected intravenously with the above-identified compounds for the purpose of determining their concentrations, respectively, in the blood. The Beagle dogs employed were of both sexes and weighed approximately 15.0 Kg. The results obtained are set forth in Table II below:
Analyzed as Z-PAM-Cl In addition to the foregoing study, the excretion (elimination) half-life (t /2) of each compound injected was determined on the basis of the blood studies obtained from Table II above. The half-life values for 2-PAM chloride and l-methyl-I,6-dihydropyridine-2- aldoxime hydrochloride are set out in Table III below.
TABLE III t kin Beagle Dogs (5 mgJKg.) Medium 2-PAM-Cl DPAM-Cl 105 minutes 168 minutes Whole Blood 150 minutes Plasma The importance of the above results shown in Tables II and III respectively, is as follows.
As indicated earlier, Z-PAM, in salt form, is a quaternary pyridinium salt, and it is therefore distributed only On the other hand, a compound of this invention, l-methyll ,6-dihydropyridine2-aldoxime hydrochloride (DPAM), which is a tertiary amine in the free-base form at the pH of blood (approximately 7.4), is distributed mainly in the blood cells and blood proteins. Consequently, the distribution ratio between the plasma versus whole blood is 1:3. In other words, twice as much of the derivative is in the blood cells and proteins than in the plasma. This assures transport of the pro-2- PAM compound through the blood-brain barrier, whereby through metabolic oxidation (described earlier), Z-PAM or a related pyridinium aldoxime can reach the brain site.
The excretion (elimination) half-life (t 56) is substantially increased in the case of DPAM. This is due to the compounds protein and blood cell binding ability. Moreover, it logically follows that DPAM is oxidized into 2-PAM before it follows the elimination pattern of 2PAM.
The result of the phenomenon observed with pro-2- PAM is that the therapeutic level of the same does not drop as fast as is the case when employing 2-PAM per se. Consequently, less frequent multiple doses will be necessary for the pyridinium aldoxime compounds of this invention as it can be expected that the t k for whole blood is somewhat longer for these compounds. It is theorized that the difference is due to the small time difference observed for the release and oxidation processes.
Urinary analysis at 12, 24 and 48 hours after parenteral administration of the pro-2-PAM compound (1- methyl- 1 ,6-dihydropyridine-2-aldoxime hydrochloride) results in a to percent recovery of the product as 2-PAM in the urine. This proves that the prodrug form is transferred into Z-PAM without any other significant secondary metabolic pathways.
Existing publications, which concern the elimination study of Z-PAM per se clearly indicate that or more percent of 2-PAM is recovered following administration, which fact clearly points in the direction of nonbody retention. See, B. V. .Iager, G. N. Stagg, and L.
Jager, Bull. Johns Hopkins H0sp., 102, 225 (1958); F.
R. Sidell andW. A. Groff, J. Pharm. Sci., 60, 124 (1971); and F. R. Sidell and A. Kaminskis, J. Pharm. Sci., 61, 1765 (1972), respectively.
Based on the above results, it can be expected that oral administration of the pyridinium aldoxime compounds of this invention will lead to at least 50 or more 13 percent absorption of same as 2-PAM versus to percent when orally administering 2-PAM per se.
As indicated earlier, when administering the pyridinium aldoxime compounds of this invention orally, they must be administered in the form of an enteric coated tablet as it has been determined that oral absorption occurs in the intestine. Therefore, any inert enteric coating can be employed so long as it will protect the pro-2-PAM compound of this invention from dissolution as it passes through the stomach and disintegrates in the small intestine. Normally, the enteric coating material is a cellulose lower fatty acid phthalate, particularly cellulose acetate phthalate. However, other cellulose derivatives can also be employed. For example, cellulose ethers or mixed ether esters can be substi' tuted for the cellulose esters. Thus, among the enteric coating materials which can be used are the materials formed by reacting cellulose acetate, cellulose propionate, cellulose acetate butyrate, ethyl cellulose butylcellulose, etc., with phthalic or maleic anhydrides or the like in the presence of a tertiary organic base. The only limitation on the enteric coating is that it shall preserve the pyridinium aldoxime compounds from dissolution until they reach the small intestine and that the enteric medium be inert (non-oxidizing). Consequently, in addition to the coating enumerated above, one can use any of the conventional enteric coatings, such as shellac and others described in that portion of REMINGTONS PRACTICE OF PHARMACY, referred to earlier in this application.
In treating the individual poisoned by an anticholinesterase blocking agent, therapeusis will be achieved, I.V. by administering about 2.5 g. to the individual initially, and then, administering one-half of that amount (1.25 g.) thereafter at 30 minute intervals until muscle strength is returned; or in the alternative, orally administering the compound of this invention in an initial dosage amount of 5.0 g. followed by the administration of the same in a dosage amount of 2.5 g. at 30 minute intervals until muscle strength return is observed.
Although the present invention has been described in great detail with reference to the specification and examples included therein, it is obviously apparent that various changes and/or modifications can be made to the same by the skilled artisan without departing from the spirit and scope thereof.
Consequently, such changes and modifications are properly, equitably and intended to be within the full range of equivalence of the following claims.
What we claim is:
l. A pro-drug compound of the pyridine aldoxime (hydroxyiminomethyl pyridinium) type capable of reactivating blocked cholinesterase having the formula:
wherein R represents a member selected from the group consisting of an alkyl (C -C group, a
group, a y
:grkNoH I: I
9 X CONH group, and a G3 -OCH N 9 X CONH whereinZ represents a member selected from the group consisting of a CH CH group, a CH- OCH group, a -Cl-l CH OCl-l Cl-l group, and a CH O-CH CH OCH group; wherein R represents a member selected from the group consisting of a hydrogen atom, a methyl group, an acyl group and a group; and wherein X represents an anion derived from a pharmaceutically acceptable acid addition salt.
2. The compound of claim 1:
l-methyl-l ,6-dihydropyridine-2-aldoxime and its HX salts, wherein X represents a pharmaceutically acceptable anion.
3. The compound of claim 1:
Trimethylene-bis-[ l-(dihydropyridine-4-carbaldoxime)] and its di-l-lX salts, wherein X represents a pharmaceutically acceptable anion.
4. The compound of claim 3:
Trimethylene-bis-[ l-( l ,4-dihydropyridine-4-carbaldoxime] and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
5. The compound of claim 3:
Trimethylene-bis-[( l ,6-dihydropyridine-4-carbaldoxime] and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
6. The compound of claim 1:
Bis-(4-hydroxyiminomethyl-dihydropyridinel methyl) ether and its di-l-[X salts, wherein X represents a pharmaceutically acceptable anion.
7. The compound of claim 6:
Bis-(4-hydroxyiminomethyl-l ,4-dihydropyridine-l methyl) ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
8. The compound of claim 6:
Bis-(4-hydroxyiminomethyl-1,6-dihydropyridine-lmethyl) ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
9. The compound of claim 1:
Bis-( 2-hydroxyiminomethyl-l ,6-dihydropyridinel methyl) ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
10. The compound of claim 1:
Bis-[2-(4-hydroxyiminomethyl-dihydropyridino) ethyl] ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
11. The compound of claim Bis-[2-(4-hydroxyiminomethyl-1,4-dihydropyridino) ethyl] ether and its di-l-lX salts, wherein X represents a pharmaceutically acceptable anion.
12. The compound of claim 10:
Bis-[2-(4-hydroxyiminomethyl-l ,6-dihydropyridino) ethyl] ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
13. The compound of claim 1:
Ethylene glycol-bis-(4-hydroxyiminomethyl-dihydropyridine-l-methyl) ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
14. The compound of claim 13:
Ethylene glycol-bis-(4-hydroxyiminomethyl-l ,4-
dihydropyridine-l-methyl) ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
15. The compound of claim 13:
Ethylene glycol-bis-(4-hydroxyiminomethyl-l ,6-
dihydropyridine-l-methyl) ether and its di-HX salts, wherein X represents a pharmaceutically acceptable anion.
16. A method for preparing a pro-drug compound of the pyridine aldoxime (hydroxyiminomethyl pyridinium) type having the formula:
wherein R represents a member selected from the group consisting of an alkyl (C -C group, a
group, and a ONH group, wherein Z represents a member selected from the group consisting of a CH CH group, a CH OCH group, a CH CH OCH CH group, and a --CH OCl-l C1-l O-CH group; wherein R represents a member selected from the group consisting of a hydrogen atom, a methyl group, an acyl group and a group; and wherein X represents an anion derived from a pharmaceutically acceptable acid addition salt, which comprises:
1. Reducing a compound of the formula:
H=NOR N l R with a suitable reducing agent selected from the group wherein Y represents said nucleophile-leaving group; 2. Preparing the HX salt of the above obtained tetrahydropyridinium compound of step (1) by introducing said compound into an ether type solvent,
saturated with anhydrous HX; 3. Eliminating an HY molecule from the HX salt of the tetrahydropyridinium compound, using a C -C aliphatic alcohol as the reaction solvent in order to obtain the objective compound wherein X and Y are defined as above; and
4. Separating the objective compound from the reac-' tion mixture, said overall reaction being conducted at standard temperature and pressure. 17. The process of claim 16, wherein in step (3) thereof, said C -C aliphatic alcohol is methanol.
18. The process of claim 16, wherein in step (1) thereof, the reducing agent is NaBH 3,929,813 17 l8 19. The process of claim 16, wherein in step (1) 21. The process of claim 16, wherein in step (2) thereof, the heterogeneous reaction mixture is an thereof, HX is HCl. ether/water mixture. 22. The process of claim 16, wherein in step (3) 20. The process of claim 16, wherein in step (2) 5 thereof, HY is HCN. thereof, the ether type solvent is tetrahydrofuran.
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|U.S. Classification||546/261, 546/338, 546/22, 546/264|
|International Classification||C07D211/82, C07D211/78|
|Cooperative Classification||C07D211/82, C07D211/78|
|European Classification||C07D211/82, C07D211/78|